The present invention relates, inter alia, to recombinant polynucleotides, proteins and methods of imparting to and/or enhancing an organisms ability to resist pathogens.
Plants resistant to pathogens often are found to evoke their resistance through a mechanism which eventually yields a hypersensitive response (HR) resulting in rapid cell death of the infected plant cells. This rapid cell death or necrosis inhibits the pathogen from further growth and thus stops the infection. This mechanism is known within the art (Klement, Z., In: Phytopathogenic Prokaryotes, Vol. 2, eds.: Mount, M. S. and Lacy, G. H., New York, Academic Press, 1982, pp. 149-177). The HR is often confused with other lesion-like phenomena, but a typical HR gives local cell death and is associated with secondary responses such as callus deposition, generation of active oxygen species, induction of phytoalexins, changes in ion fluxes across membranes and induction of acquired resistance (AR) (Hammond-Kosack, K. E., et al., Plant Physiol. 110, 1381-1394, 1996).
Pathogen resistance can be elicited by response to elicitor compounds, which are frequently found to be of proteinaceous nature (Arlat, M., et al., EMBO J., 13, 543-553, 1994; Baker, C. J. et al., Plant Physiol. 102, 1341-1344, 1993; Staskawicz, B. J. et al., Proc. Natl. Acad. Sci. USA 81, 6024-6028, 1984; Vivian, A. et al., Physiol. Mol. Plant Pathol. 35, 335-344, 1989; Keen, N. T., Ann. Rev. Gen. 24, 447-463, 1990; Ronald, P. C. et al., J. Bacteriol. 174, 1604-1611, 1992; Whithan, S. et al., Cell 78, 1-20, 1994; Kobe, B. and Deisenhofer, J., Trends Biochem. Sci. 19, 415, 1994; and Honxc3xa9e G. et al., Plant Mol. Biol. 29, 909-920, 1995). These elicitor proteins (encoded by avirulence genes) are thought to bind to a resistance protein available in the plant, therewith starting a cascade of events resulting in the HR. The elicitor proteins are characterized by the fact that they are race-specific and only are able to elicit the response with a corresponding (also specific) resistance protein. The concept of avirulence-gene based resistance is also known under the name of the gene-for-gene response. Avirulence genes have been cloned from bacterial pathogens (such as Pseudomonas and Xanthomonas) and from fungal pathogens (such as Cladosporium fulvum, Rhynchosporium secalis and Phytophthora parasitica). Also plant genes coding for some of the corresponding resistance genes have been cloned (such as the tomato gene Cf9 corresponding to the avirulence gene avr9 from Cladosporium fulvum, and the tomato Pto-gene corresponding to the avirulence gene avrPto from Pseudomonas).
It has recently become clear that the plant resistance proteins when activated by interaction with the pathogen-derived elicitor proteins are capable of inducing a signal transduction pathway. It has been established that some interactions at least partly use a common pathway (Century, K. S., et al., Science 278, 1963-1965, 1997). In this publication the NDR1 locus has been shown to be required for resistance to the bacterial pathogen Pseudomonas syringae pv. tomato and to the fungal pathogen Peronospora parasitica. Similarly Parker, J. E., et al. (The Plant Cell 8, 2033-2046, 1996) have shown that the product encoded by the eds1-locus in Arabidopsis thaliana also has a key function in the signal transduction pathway after infection with Peronospora parasitica, but not after infection with Pseudomonas syringae pv glycinae.
Recently a report has been published stating that also plant derived proteins can elicit cell-death like phenomena (Karrer, E. E. et al., Plant Mol. Biol. 36, 681-690, 1998). In this publication 11 clones have been described which were able to produce lesions in tobacco plants.
Methods to use resistance genes to confer pathogen resistance to plants are often hampered by the fact that the resistance is only limited to a few specific pathotypes.
Thus there is still need for a system which can convert a general pathogen resistance to plants which is silent when no pathogens are infecting.
The invention now concerns a method for the induction of pathogen resistance in plants characterized by transforming, a plant with a polynucleotide sequence comprising a pathogen inducible promoter which regulates the expression of a plant signal transduction protein or a homologue thereof which when expressed gives rise to a hypersensitive response in plants.
Specifically the signal transducing protein is ndr1 or a homologue thereof, eds1 or a homologue thereof, or Xa21 or a homologue thereof.
It is also possible that the signal transduction protein is selected from the group consisting of a G-protein, a protein kinase, an AMP-cyclase and a protein phosphatase. Another embodiment of this part of the invention is where the signal transduction protein is a mutant from a signal transduction protein, which, when expressed, yields a hypersensitive response. These mutants preferably are encoded on the acd locus or the lsd locus. Specifically mutants would be ndr1-PKC, ndr1-cDPK and truncated Xa21.
An other embodiment of the invention is a method for the induction of pathogen resistance in plants characterized by transforming a plant with a polynucleotide sequence comprising a pathogen inducible promoter which regulates the expression of a compound which is able to alleviate the inhibitory effect of a protein on the signal transduction pathway leading to a hypersensitive response in plants. An example of such a method is a method wherein the compound is an mRNA which is coding for the inhibitory protein in an anti-sense orientation, a method wherein the compound is interacting sterically with the inhibitory protein or a method wherein the compound is an antibody, a ribozyme or an RNA molecule which is able to suppress translation of the mRNA coding for the inhibitory protein.
Also part of the invention are polynucleotides comprising a pathogen inducible promoter sequence operably linked to a protein encoding sequence which encodes a plant signal transduction protein which is active in the signal transduction pathway of a plants hypersensitive response. Preferably these transduction proteins are selected from the group comprising of ndr1, eds1 and Xa21. More generally, the signal transduction proteins are selected from the group consisting of a G-protein, a protein kinase, an AMP-cyclase and a protein phosphatase. Another example of these signal transduction proteins are mutants from a signal transduction protein, which when expressed gives rise to a hypersensitive response in plants, especially those where the mutant protein is encoded on the acd locus or the lsd locus. Specifically preferred mutants are selected from the group of ndr1-PKC, ndr1-cDPK and truncated Xa21.
Yet another embodiment of the invention are polynucleotides comprising a pathogen inducible promoter sequence operably linked to a sequence which is able to alleviate the inhibitory effect of a protein which is active in the signal transduction pathway of a plants hypersensitive response.
The pathogen-inducible promoters for these polynucleotides can be selected from the group comprising of the promoters of prp1, Fis1, Bet v 1, Vst1, gstA1, and sesquiterpene cyclase, but any pathogen-inducible promoter which is switched on after pathogen infection can be used.
Also part of the invention is a method using a polynucleotide as described above to transform a plant, making said plant at least partially resistant to pathogens.